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Journal of Biomolecular Screening
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Simulation Modeling of Pooling for Combinatorial Protein Engineering

Karen M. Polizzi

School of Chemical & Biomolecular Engineering

Cody U. Spencer

College of Computing, Georgia Institute of Technology, Atlanta, GA

Anshul Dubey

School of Chemical & Biomolecular Engineering

Ichiro Matsumura

Department of Biochemistry, School of Medicine, Emory University, Atlanta, GA

Jay H. Lee

Matthew J. Realff

Andreas S. Bommarius

School of Chemical & Biomolecular Engineering

Pooling in directed-evolution experiments will greatly increase the throughput of screening systems, but important parameters such as the number of good mutants created and the activity level increase of the good mutants will depend highly on the protein being engineered. The authors developed and validated a Monte Carlo simulation model of pooling that allows the testing of various scenarios in silico before starting experimentation. Using a simplified test system of 2 enzymes, ßgalactosidase (supermutant, or greatly improved enzyme) and •-glucuronidase (dud, or enzyme with ancestral level of activity), themodel accurately predicted the number of supermutants detected in experimentswithin a factor of 2. Additional simulations usingmore complex activity distributions showthe versatility of themodel. Pooling ismost suited to cases such as the directed evolution of newfunction in a protein, where the background level of activity is minimized, making it easier to detect small increases in activity level. Pooling ismost successful when a sensitive assay is employed. Using the modelwill increase the throughput of screening procedures for directed-evolution experiments and thus lead to speedier engineering of proteins.

Key Words: directed evolution • high-throughput screening • Monte Carlo simulation • protein engineering

This version was published on December 1, 2005

Journal of Biomolecular Screening, Vol. 10, No. 8, 856-864 (2005)
DOI: 10.1177/1087057105280134
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